A minimally invasive, scalable and reproducible neonatal rat model of severe focal brain injury

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Abstract
Neonatal brain injuries, such as stroke, cause focal ischaemic lesions that often result in lifelong neurological disabilities, yet effective treatments remain limited. Early-phase therapeutic screening requires models that can reliably reproduce injury severity while minimising confounding variables, including prolonged or variable anaesthesia, surgical stress, and invasive procedures that themselves affect injury progression. Existing models of neonatal focal ischaemia often exhibit high mortality, technical complexity, and substantial variability in lesion location and volume. As a result, there is a critical need for a rapid, ethically refined, and scalable neonatal model that produces consistent cortical injury suitable for screening neuroprotective, biomaterial-based, and regenerative therapies. We established a minimally invasive photothrombotic ischaemia model in postnatal day 10 rats by administering intraperitoneal Rose Bengal (25, 40, or 60 mg/kg) and activating it with a fixed 10-minute exposure to 565-nm light through the intact scalp and skull. This incision-free protocol allowed a total procedure duration of 19 min. We characterized dose-dependent effects on infarct volume and anatomical distribution, cortical atrophy, ventricular enlargement, apoptosis (cleaved caspase-3), astrocytic and microglial reactivity (glial fibrillary acidic protein, GFAP; ionized calcium-binding adapter molecule 1, Iba1), and sensorimotor outcomes (wire hang, cylinder rearing, adhesive tape removal) at 1, 7, and 14 days after injury. Additional analyses assessed the reproducibility of lesion size across litters and explored sex-specific differences. A 25 mg/kg dose induced a reliable and well-localized motor cortex infarct with no mortality. Higher doses of Rose Bengal produced proportionally larger infarcts with greater subcortical involvement and more pronounced secondary atrophy. Across all groups, apoptotic signalling and glial reactivity remained elevated through 14 days, indicating persistent tissue injury. Sensorimotor impairments were robust at all stages, with deficits in forepaw use, endurance, and tactile response correlating with lesion volumes in the 25 mg/kg group. No significant sex differences were observed for any histological or behavioural outcomes. This refined neonatal photothrombotic model provides a reproducible, simple, scalable, and ethically optimized platform for inducing severe focal cortical injury. The model’s stable injury territory, short, standardized procedure, and consistent functional readouts fill a major gap in current research tools and provide a practical foundation for early-phase testing of neuroprotective and regenerative interventions.
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